PROJECT SUMMARY Human astroviruses (HAstV) are a leading cause of viral diarrhea in children. Divergent HAstV strains are associated with fatal human encephalitis. Currently, no vaccines or antiviral therapies exist for HAstV infections. Our goal is to identify sites of vulnerability (Achilles' heels) on the HAstV surface that can be exploited for the development of preventative and therapeutic strategies against HAstV. The HAstV virion is a small, icosahedral virus composed of an RNA genome surrounded by capsid protein. Despite the important role of the HAstV capsid protein in virus entry into host cells, the location of antibody epitopes and the mechanisms of virus-neutralizing antibodies are unknown. Our central hypothesis is that the HAstV capsid spike domain plays a key role in virus entry and is a target of HAstV-neutralizing antibodies. Using a diverse toolbox of structural, biochemical, immunological, and virological approaches, we will pursue three specific aims to (1) use structural and biophysical studies to define HAstV capsid epitopes and serotype-reactivity of a panel of six neutralizing monoclonal antibodies, (2) determine neutralizing mechanisms of a panel of neutralizing monoclonal antibodies targeting the HAstV capsid spike, and define the role of the HAstV capsid spike in cell entry, and (3) determine the structures of the divergent, neurotropic HAstV-VA1 capsid core and spike, test their abilities to elicit neutralizing antibodies, and define HAstV-VA1 neutralizing epitopes. This work represents the first molecular study of HAstV neutralizing epitopes. Results obtained by this work will elucidate mechanisms of HAstV entry and neutralization and provide a foundation for the design of vaccines and therapeutics to prevent and treat HAstV infections.